The miR-26 family regulates early B cell development and transformation

We show that the activity of the miR-26 family determines early B cell behavior: high miR-26 levels promote cell expansion and block the pre-B to immature B cell transition, whereas a miR-26 reduction limits expansion and enhances pre-B cell differentiation.

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Overall, the manuscript is compelling and appropriate for LSA and will be of interest to a broad audience, including immunologists and cancer biologists. There are a few points that should be addressed: 1. There is a prior publication that hints at the role of miR-26 in the regulation of PI3K signaling/Pten -a Nature Imm report from 2016 by Gonzalez-Martin et al. shows that miR-26a targets PTEN in WEHI-231 B cell line. While the present manuscript goes into much more mechanistic detail and provides much novel insight, the 2016 publication should be appropriately acknowledged.
2. In vivo studies, such as those presented in Fig. 6 should include absolute counts, and not just relative % for B cell subsets to substantiate the statements about the impact of miR-26 on enhancing/accelerating differentiation of pre-B cells. Fig. 1 -is there any direct data on proliferation? The authors make a point of augmented proliferation being responsible for the phenotype solely based on lack of impact on survival, perhaps a direct assessment of proliferation would let them make a stronger point. 4. In systems where miR-26 is overexpressed and therefore PI3K signaling is augmented, is there any evidence of Ig deficient B cells persisting in the periphery -as BCR tonic signal is dependent on PI3K, perhaps some of the accelerated differentiation is due to this? 5. Can the lack of evident difference in PTEN (or really other readily recognizable miR-26 targets) in Fig 7 be due to the cells simply not tolerating high PTEN expression and subsequent diminished PI3K signaling?

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Reviewer #2 (Comments to the Authors (Required)): The manuscript of Hutter et al. on "The miR-26 family regulates early B cell development and transformation" studies the function and the role of the small micro-RNA miR-26a in early B cell development. The starting point of this study was a screen for micro RNAs which blocked the transition from pre-B cells to immature B cells induced by IL-7 withdrawal. In this screen the authors show that overexpression of miR-26 blocks B cell maturation and they confirm this result in a more detailed analysis. In a search for pre-B cell expressed genes, which are affected by overexpression of miR-26, the authors identify, among others, PTEN as a candidate which is regulated by this micro-RNA. It is well known that PI3 kinase signaling is promoting pre-B cell expansion and that the switch from pre-B cell to immature B cells is accompanied by reduced PI3 kinase signaling and increased expression of PTEN, a lipid phosphatase that inhibits the PI3 kinase signaling pathway. Thus, the identification of PTEN as a target gene for miR-26 fits to the observed phenotype. However, given the fact that the reduction of PTEN expression is variable, it is not completely clear whether PTEN is the dominant miR-26 target or whether other genes also are affected by overexpression of miR-26 or vice versa. I therefore think it would be important to show whether or not an overexpressing of PTEN to different levels, counteracts the blocking effect of miR-26 in the pre-B to immature B cell transition. Otherwise, this study is interesting and worthwhile to be published in the Life Alliance Journal.
1. In their study of miR-26 function the authors are using different pre-B cell lines or primary B cell progenitors, but it is not always clear what lines are used. Thus, it would be helpful for the reader if the authors indicate the use of the cells not only in the figure legends, but also in the figure itself. Furthermore, what does it mean when they write in the figure legend, for example under Fig. 1c they use Wk3 pre-B cells or primary B cell progenitors? For which figure, Fig. 1D or 1E do the authors use primary B cell progenitors? Furthermore, it would be important that the authors state why they do some experiments with 1676 pre-B cell line and others with the Wk3 line. 2. The survival and anti-apoptosis effect of miR-26 is shown in Fig 1 E and F. Fig 1E shows the pro-survival effect mediated by miR-26. Both results {plus minus} miR-26 are within the normal apoptosis range of cultivated cell lines, so it would be desirable to repeat this experiment (N=?) and perform a significance calculation.For the calculation of the living cell population in Fig 1F, it should be shown, how the gates were set in the FACS plot. 3. The anti phospho-tyrosine blot shown in Fig. 2C is not very informative without knowing what phosphorylated substrates are analyzed in this blot. The direct connection between miR-26 expression and PTEN is also not clear if one regards the result of the removal of the miR-26 by a specific sponge where the PTEN expression is not really increased. Although the introduced sponge clearly increases the pre-B cell to immature B cell transition, these data suggest that also other genes (or other mi-Rs e.g., miR-19, miR-19~92, miR-150, please comment) are involved in the regulation of this process and it would be helpful to learn more about those, if possible. In the differentiation studies shown in Fig. 6C it would be interesting whether the authors extend their studies not only on the IgM-BCR, but also on the IgD-BCR and the lambda light chain isotype. 4. The authors stated in their discussion:" miR-26-mediated PTEN downregulation has been reported for T cell acute lymphoblastic leukemia (T ALL) and lung cancer". It would be interesting how they explain the fact that miR-26a was found down-regulated in all patients with B-ALL (Cancer Biomark. 2015;15(3):299-310. doi: 10.3233/CBM-150465) when they relate the findings to pre-B cells leukemia.
Minor points: 1. In the introduction the authors use the term "light chain recombination" or rearrangement of light chain gene segments. I think they should here stick to the proper nomenclature. There is no rearrangement of light chains, but of light chain variable genes. Furthermore, the rearrangement process involves V gene segments and not light chain gene segments.
2. Fig 5 A shows that inhibition with miR-26 sponge increases the population of differentiated cells. The number of cells measured in miR-26 sponge/dsRed+ seems to be significantly higher than in the other gates. Although the percentage distribution would be unaffected, it is more reliable to measure comparable cell numbers. 3. Some experiments and data are "not shown". It would be desirable to include them in the supplemental part. 4. K.K. and S.H. wrote .... Please correct author contributions Dear Editors, dear Reviewers, we would like to thank you for the time you invested into critically reading and evaluating our manuscript "The miR-26 family regulates early B cell development and transformation", which we have submitted for publication as an article in Life Science Alliance. We appreciate the positive comments about our work and the insightful suggestions that enabled us to improve the quality of our manuscript.
Although not requested by the Reviewers, please note that in the revised manuscript we have provided quantitative PCR data showing that pre-B cells expressing miR-26 fail to induce critical genes such as Rag1, Rag2 and Aiolos upon IL-7 withdrawal (suppl. Fig.  1A), which corroborates the block in differentiation as measured by kappa light chain expression.
That said, in the following pages we address each concern raised by the Reviewers point by point.

Reviewer 1
Comment: There is a prior publication that hints at the role of miR-26 in the regulation of PI3K signaling/Pten -a Nature Imm report from 2016 by Gonzalez-Martin et al. shows that miR-26a targets PTEN in WEHI-231 B cell line. While the present manuscript goes into much more mechanistic detail and provides much novel insight, the 2016 publication should be appropriately acknowledged.
Response: We thank this Reviewer for this suggestion. Indeed, while the key finding of this study is the identification of miR-148 as a critical regulator of central tolerance, the experimental approach with miRNA pools that were used to transduce HSCs also retrieved miR-26a and b as tolerance breaking. As this Reviewer pointed out, in the supplementary data the authors show that overexpression of miR-26a results in repression of PTEN in WEHI-231 cells, which is in accordance with our own findings. To acknowledge this study, we have included its reference in the discussion.
Comment: In vivo studies, such as those presented in Fig. 6 should include absolute counts, and not just relative % for B cell subsets to substantiate the statements about the impact of miR-26 on enhancing/accelerating differentiation of pre-B cells.
Response: We agree with this Reviewer that absolute cell counts in addition to relative percentages should in general be provided with all in vivo experiments. In this particular case, however, we would like to point out that only a subset of cells within our miR-26 sponge model and the respective control express the transgene (see also Figs. 6B and C). Moreover, the scrambled sponge mouse displays significantly higher portion of GFP + cells  Fig. 6B to D. If this Reviewer thinks that such a graph would be beneficial for our manuscript, we are of course happy to add it to the main figure or as a supplement. Fig. 1 -is there any direct data on proliferation? The authors make a point of augmented proliferation being responsible for the phenotype solely based on lack of impact on survival, perhaps a direct assessment of proliferation would let them make a stronger point.

Reviewer 2
Comment Response: We apologize for the unclear labeling of the figures. As suggested, we now provide labels for all figures, and state more clearly in the figure legends which type of cells were used in the respective panel. In case of Figure 1C to E, the primary data (upper panels in the respective figure) were generated in 1676 cells, but the same type of experiment was repeated with primary, bone marrow-derived pro-/pre-B cells and the statistical analysis of these experiments is provided in the bar graphs below. Of note, Fig. 1C Fig 1F, it should be shown, how the gates were set in the FACS plot.
Response: We agree with this reviewer that total effect on apoptosis was weak in the experiments shown in Fig. 1E. However, the relative protective effect mediated by miR-26 was nevertheless clearly significant (see bar graph below the primary data in the original figure; n=8). Still, we decided to repeat these experiments with a freshly thawed batch of cells and now show a higher basal apoptosis rate upon IL-7 withdrawal, but the same or an even stronger pro-survival effect by miR-26 (Fig. 1E).
As requested by this Reviewer, we have furthermore added the gates to the FSC-SSC plots in Fig. 1F.
Comment: The anti phospho-tyrosine blot shown in Fig. 2C is not very informative without knowing what phosphorylated substrates are analyzed in this blot. The direct connection between miR-26 expression and PTEN is also not clear if one regards the result of the removal of the miR-26 by a specific sponge where the PTEN expression is not really increased. Although the introduced sponge clearly increases the pre-B cell to immature B cell transition, these data suggest that also other genes (or other mi-Rs e.g., miR-19, miR-19~92, miR-150, please comment) are involved in the regulation of this process and it would be helpful to learn more about those, if possible. In the differentiation studies shown in Fig.  6C it would be interesting whether the authors extend their studies not only on the IgM-BCR, but also on the IgD-BCR and the lambda light chain isotype.
Response: We agree that it would have been useful to identify the differentially phosphorylated proteins in cells undergoing pre-B to immature B cell differentiation. However, we would like to emphasize that the anti-phosphotyrosine antibody (clone 4G10) used here is well known for its performance, whereas specific anti-phosphotyrosine antibodies often generate only weak signals, in particular under steady state conditions. Thus, the most suitable experimental strategy in this case would have been a mass spectrometric approach, but we think that this is a separate project and beyond the scope of this manuscript. The purpose of this blot was simply to illustrate that the "pattern" of intracellular signaling associated with IL-7 withdrawal, which is likely dominated by signals from the IL-7R and the pre-BCR, is counteracted by miR-26 expression. Clearly, Fig. 2C is not a key figure for the overall storyline, and we are happy to move it to the supplements or to remove it completely if this is requested. Regarding PTEN and in contrast to the gain-of-function situation, we agree that the data presented do not necessarily support this critical signaling regulator as a putative target in the loss-of-function setting. As we discuss, we hypothesize that the loss-of-function phenotype is established by subtle changes of several genes, possibly including PTEN, but that this may not be identified by our "global" approaches due to large cell-to-cell variation or overall subtle but still functional effects. Alternatively, we speculate that miR-26 may directly co-regulate additional genes that are counteracted by PI3K signaling, and that may be implicated in the pre-B to immature B cell transition. This would explain why PTEN knockdown recapitulates the miR-26a overexpression effects while at the same time the loss of miR-26 function does establish the reciprocal phenotype apparently independent of PTEN. However, we have not identified such genes in the gain-or in the loss-of-function microarray analysis.
Regarding other coding and non-coding genes involved in early B cell development, we certainly agree that miRNAs of the miR-17-92 cluster are implicated in pro-and possibly also pre-B cell survival and function (Ventura et al., Cell 2008;Lai et al., Nat. Comm. 2016). In fact, our own work using a miRNA sponge library (based on the same principles as shown for miR-26 in this study) has revealed a putative role for the miR-15 family, but also for the miR-17 (miR-17 and miR-20), miR-19 (miR-19a and miR-19b), and miR-25 (miR-25 and miR-92) families in the pre-B to immature B cell transition (Lindner et al., EMBO Rep. 2017

Minor points:
Comment: In the introduction the authors use the term "light chain recombination" or rearrangement of light chain gene segments. I think they should here stick to the proper nomenclature. There is no rearrangement of light chains, but of light chain variable genes. Furthermore, the rearrangement process involves V gene segments and not light chain gene segments.
Response: We apologize for these imprecise terms and have corrected the respective sections throughout the manuscript.
Comment: Fig 5 A shows that inhibition with miR-26 sponge increases the population of differentiated cells. The number of cells measured in miR-26 sponge/dsRed+ seems to be significantly higher than in the other gates. Although the percentage distribution would be unaffected, it is more reliable to measure comparable cell numbers.
Response: We agree that it would be optimal to compare similar cell numbers in the dsRedand the dsRed+ populations of one sample. In this particular case (Fig. 5A), the contour plot appears misleading, as the dsRed+ percentage distribution was 16 % for the scrambled sponge and 8 % for the miR-26 sponge, respectively. Thus, in the latter sample the dsRedpopulation was actually much higher than the dsRed+ population, in contrast to the visual impression. In this context, we would like to emphasize that these percentages depend on the transduction efficiency and thus on the virus titer, which often cannot be precisely adjusted to give rise to a 50:50 ratio. In this particular experiment (n=15 for the 1676 cells), we had a broad range of dsRed+ percentages in the replicates, but this did not alter the biological effect. We consider this as strong evidence that the percentage distribution is not affected by differences in transduction efficiencies, as this Reviewer itself points out.
Comment: Some experiments and data are "not shown". It would be desirable to include them in the supplemental part. We do not show, however, data where we found no transforming activity, such as in of miR-26a in bone marrow-derived primary B cell precursors expressing miR-26a as well as upon PTEN knockout or myristoylated Akt expression in 1676 cells. These experiments would resemble Figure 2A, except that all cells die out over time. We think that such a data panel would be of limited interest for the readers, and hope that this Reviewer agrees on this.